Liver funcion protecting or ameliorating agent

ABSTRACT

A liver function protecting or improving agent which comprises a compound represented by the formula (I)  
                 
 
     {in the formula (I), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  may be the same or different, and represent hydrogen, halogen, hydroxy, alkoxy or alkyl; and R A  represents the formula (II)  
                 
 
     [in the formula (II), R 10  and R 11  may be the same or different, and represent hydrogen or halogen, or R 10  and R 11  together represent a binding] or the formula (III)  
                 
 
     [in the formula (III), R 12  represents hydrogen, halogen, hydroxy, alkoxy, cyano or alkyl, R 13  and R 14  may be the same or different, and represent hydrogen or halogen, or R 13  and R 14  together represent a binding]} or a glycoside thereof or a pharmaceutically acceptable salt thereof.

TECHNICAL FIELD

[0001] The present invention relates to a liver function protecting orimproving agent, a food and drink or a feed for protecting or improvingliver functions, and an additive for foods and drinks or additives forfeeds having liver function protecting or improving activity.

BACKGROUND OF THE INVENTION

[0002] The liver is an important organ which has various functions suchas metabolic regulation and storage of sugar, protein and lipid whichare three major nutrients, and decomposition and detoxification ofsubstances unnecessary to the living body. These functions suffer acuteor chronic disorders due to an excessive in take of alcohol, viralinfection, bad eating habits, stress, smoking, etc. The advance of thesedisorders results in diseases such as acute hepatitis, chronichepatitis, hepatic cirrhosis, alcoholic fatty liver, hepatitis B andliver cancer.

[0003] When liver cells are damaged by virus, alcohol, etc., enzymessuch as aspartate aminotransferase (glutamic-oxaloacetic transaminase,hereinafter abbreviated as GOT) and alanine aminotransferase(glutamic-pyruvic transaminase, hereinafter abbreviated as GPT) in thecells leak into the blood, which raise the values indicating theactivities of these enzymes. Accordingly, the levels of GOT and GPTactivities in the blood are known as indices of the levels of the liverfunction disorders.

[0004] Known drugs used for the prevention or treatment of the liverfunction disorders include antiviral agents such as acyclovir,immunosuppressive agents, glutathione and the like. Foods and drinkswhich are recognized to be effective for protecting, strengthening andimproving the liver functions include for example, turmeric, milkthistle, sesame lignan, oyster extract, liver extract and the like.

[0005] However, a strong need consistently exists for the development ofpharmaceutical agents which are effective in prevention or treatment ofliver diseases, and of health foods and drinks or animal feeds whichenable prevention or treatment of hepatopathy by daily intake.

[0006] In connection with stilbenoid compounds, anti-allergicactivities, anti-oxidative activities and anti-bacterial activities havebeen conventionally investigated on compounds isolated from HydrangeaeDulcis Folium which is a herbal medicine [Summary of Lectures at the 2ndSymposium on Medicines and Foods, p. 85, 1999, Nihon Yakuyou ShokuhinGakkai Zyunbi Iinkai (Japanese Society of Medicated Foods, PreparatoryCommittee)], and the like.

[0007] Reports have been made as follows on in vitro activities of thestilbenoid compounds.

[0008] In regard to the anti-allergic activities, it is reported thatrelease of histamine from mast cells induced by compound 48/80 orcalcium ionophore A23187 is suppressed by thunberginol A-G (50%inhibitory concentration: 9.4-92 μM), but is not suppressed byphyllodulcin or phyllodulcin 8-O-glucoside, hydrangenol, hydrangenol8-O-glucoside, hydramacrophyllol A and B at 100 μM [Bioorganic &Medicinal Chemistry, 7, 1445 (1999)].

[0009] The extract of Hydrangeae Dulcis Folium (2000 mg/kg) is reportedto suppress rat skin passive anaphylaxis through oral administration[Journal of the Pharmaceutical Society of Japan, 114(6), 401 (1994)]. Inaddition, it is reported that phyllodulcin 8-O-glucoside (300 and 500mg/kg), hydrangenol (500 mg/kg), hydrangenol 8-O-glucoside (300 and 500mg/kg), thunberginol A (300 and 500 mg/kg), thunberginol F (300 and 500mg/kg) suppress rat skin passive anaphylaxis through oraladministration, but phyllodulcin (300 and 500 mg/kg) does not suppressrat skin passive anaphylatic reaction [Biological & PharmaceuticalBulletin, 22(8), 870 (1999)].

[0010] In connection with the anti-oxidative activities, it is reportedthat: as to DPPH (1,1-Diphenyl 2-Picrylhydrazyl) radical capturingcapacity, the extract of Hydrangeae Dulcis Folium exhibits 50% capturingaction at 0.099 mg, while phyllodulcin exhibits this activity at 0.208mg, and hydrangenol at 1.074 mg; as to linoleic acid oxidation (ironrhodanate method), phyllodulcin exhibits more potent suppressiveactivity than α-tocopherol or BHA (Butylated Hydroxyanisole), whilehydrangenol exhibits a weak suppressive activity thereto; and as to NADP(Nicotinamide Adenine Dinucleotide Phosphate) H dependent lipidperoxidation in rat liver microsome, phyllodulcin exhibits a suppressiveactivity at a similar level to α-tocopherol, while hydrangenol exhibitsa weak suppressive activity thereto [Natural Medicine, 49(1), 84(1995)].

[0011] In regard to anti-bacterial activity, it is reported thatphyllodulcin at 1000 ppm exhibits a proliferation suppressive activityon Staphylococcus aureus and Staphylococcus epidemidis (JapanesePublished Unexamined Patent Application No. 43460/93), and phyllodulcinand hydrangenol exhibit an anti-bacterial activity on Bacteroidesmelaninogenicus and Fusobacterium nucleatum with the minimum growthinhibitory concentration of 1.25-2.5 ppm (Japanese Published UnexaminedPatent Application No. 92829/94).

[0012] In regard to differentiation-inducing activity on leukocyte[Chemical & Pharmaceutical Bulletin, 48 (4), 566 (2000)], isocoumarinssuch as thunberginol A (active at 100 μM) are reported to have morepotent activity in vitro than dihydroisocoumarin such as phyllodulcin orhydrangenol (active at 300 μM). In addition, hydrangenol is reported tohave a hyaluronidase inhibitory activity [Planta Medica, 54, 385(1988)].

[0013] Moreover, it is reported that the extract of Hydrangeae DulcisFolium (500 mg/kg) exhibits a cholagogic activity in an in vitro test,however, phyllodulcin (200 mg/kg) and hydrangenol (200 mg/kg) do notexhibit this activity [Journal of the Pharmaceutical Society of Japan,114 (6), 401 (1994)].

[0014] Furthermore, it is reported that the extract of Hydrangeae DulcisFolium (200 and 400 mg/kg, oral administration) suppresses rat gastriculcer induced by ethanol hydrochloride, however, phyllodulcin (75 mg/kg,oral administration) and hydrangenol (75 mg/kg, oral administration) donot suppress the ulcer [Journal of the Pharmaceutical Society of Japan,114(6), 401 (1994)]

DISCLOSURE OF THE INVENTION

[0015] An object of the present invention is to provide a liver functionprotecting or improving agent, a food and drink or a feed for protectingor improving liver functions, and an additive for foods and drinks or anadditive for feeds for protecting or improving liver functions.

[0016] The present invention relates to the following (1) to (15).

[0017] (1) A liver function protecting or improving agent whichcomprises a compound represented by the formula (I)

[0018] {in the formula (I), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ may bethe same or different, and represent hydrogen, halogen, hydroxy, alkoxyor alkyl; and R^(A) represents the formula (II)

[0019] [in the formula (II), R¹⁰ and R¹¹ may be the same or different,and represent hydrogen or halogen, or R¹⁰ and R¹¹ together represent abinding] or the formula (III)

[0020] [in the formula (III), R¹² represents hydrogen, halogen, hydroxy,alkoxy, cyano or alkyl, R¹³ and R¹⁴ may be the same or different, andrepresent hydrogen or halogen, or R¹³ and R¹⁴ together represent abinding]} or a glycoside thereof [hereinafter referred to as compound(I)] or a pharmaceutically acceptable salt thereof.

[0021] (2) The liver function protecting or improving agent according to(1), wherein R¹, R³, R⁵, R⁸ and R⁹ represent hydrogen.

[0022] (3) The liver function protecting or improving agent according to(1) or (2), wherein R² represents hydrogen or hydroxy, R⁴ representshydroxy, R⁶ and R⁷ may be the same or different, and represent hydrogen,hydroxy or alkoxy.

[0023] (4) The liver function protecting or improving agent according toany one of (1) to (3), wherein R^(A) represents the formula (II).

[0024] (5) The liver function protecting or improving agent according toany one of (1) to (3), wherein R^(A) represents the formula (III).

[0025] (6) The liver function protecting or improving agent according to(1), wherein the compound (I) is phyllodulcin.

[0026] (7) The liver function protecting or improving agent according to(1), wherein the compound (I) is hydrangenol.

[0027] (8) A food and drink in which the liver function protecting orimproving agent according to any one of (1) to (7) is added.

[0028] (9) A feed in which the liver function protecting or improvingagent according to any one of (1) to (7) is added.

[0029] (10) An additive for foods and drinks in which the liver functionprotecting or improving agent according to any one of (1) to (7) isadded.

[0030] (11) A feed additive in which the liver function protecting orimproving agent according to any one of (1) to (7) is added.

[0031] (12) The liver function protecting or improving agent accordingto anyone of (1) to (7), which is administered orally.

[0032] (13) The liver function protecting or improving agent accordingto any one of (1) to (7), wherein the liver function is a functionaffected by an alcohol.

[0033] (14) A method for protecting or improving a liver function whichcomprises administering a therapeutically effective amount of thecompound (I) or a pharmaceutically acceptable salt thereof to an animalincluding human.

[0034] (15) Use of the compound (I) or a pharmaceutically acceptablesalt thereof for manufacturing a liver function protecting or improvingagent.

[0035] In the definition of each group in the compound (I), the alkylmoiety of the alkyl and alkoxy may be for example, straight chain orbranched alkyl having 1 to 10 carbon atoms, more preferably having 1 to6 carbon atoms. More specifically, examples include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,neopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like. Halogenrepresents each atom of fluorine, chlorine, bromine or iodine.

[0036] Kind and the chain length of the sugar that constitutes theglycoside are not particularly limited. Examples of the kind of thesugar include pentose, hexose, heptose and the like, and hexose ispreferred. Examples of pentose include D-xylose, L-arabinose,D-arabinose, D-ribulose, D-xylulose, L-xylulose, D-ribose, D-deoxyriboseand the like. Examples of hexose include D-glucose, D-fructose,D-mannose, D-galactose, L-galactose, D-tagatose, L-sorbose, L-fucose,D-fucose, D-quinovose, L-rhamnose and the like. Examples of heptoseinclude sedoheptulose, persulose and the like.

[0037] The chain length of the sugar is for example, 1 to 10, preferably1 to 6, and more preferably 1 to 3.

[0038] Examples of the pharmaceutically acceptable salt of the compound(I) include salts with for example, inorganic acids (e.g., hydrochloricacid, sulfuric acid, nitric acid and the like), organic acids (e.g.,carbonic acid, bicarbonic acid, succinic acid, acetic acid, propionicacid, trifluoroacetic acid and the like), inorganic bases (e.g., alkalimetals such as sodium or potassium, alkaline earth metals such ascalcium or magnesium, and the like) and organic base compounds (e.g.,organic amines such as triethylamine, basic amino acids such asarginine, and the like), and the like.

[0039] Examples of the compound (I) include compounds represented by theformula (IV)

[0040] [in the formula (IV), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ andR¹¹ each are as defined previously] or glycosides thereof, or compoundsrepresented by the formula (V)

[0041] [in the formula (V), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³and R¹⁴ each are as defined previously] or glycosides thereof.

[0042] In the formula (I), formula (IV) and formula (V), R¹, R³, R⁵, R⁸and R⁹ are preferably hydrogen. Furthermore, in the formula (I), formula(IV) and formula (V), R² is preferably hydrogen or hydroxy, R⁴ ispreferably hydroxy, R⁶ and R⁷ are the same or different, and arepreferably hydrogen, hydroxy, or alkoxy.

[0043] Specific examples of the compound (I) include phyllodulcin,hydrangenol, thunberginol A, phyllodulcin 8-O-glucoside, phyllodulcin3′-O-glucoside, hydrangenol 8-O-glucoside, hydrangenol 4′-O-glucoside,3-(fluorophenyl)-3,4-dihydroisocoumarin, 3-(fluorophenyl)isocoumarin,3-(3′,5′-dimethyl-4′-hydroxybenzylidene)phthalide,4,5,6,7-tetraiodo-3-benzalphthalide,4,5,6,7-tetraiodo-3-(α-cyanobenzal)phthalide,8-methoxy-3-(p-methoxyphenyl)isocoumarin,3,4-dihydro-3-(p-methoxyphenyl)isocoumarin,5-chloro-8-hydroxy-6-methoxy-3-phenylisocoumarin and the like. Thesestructures are show in Table 1. TABLE 1

[0044] The compound (I) or a pharmaceutically acceptable salt thereofmay be chemically synthesized, or may be isolated and purified from anatural product which originally contains the compound (I) or apharmaceutically acceptable salt thereof.

[0045] Illustrative examples of the process for chemically synthesizingthe compound (I) or a pharmaceutically acceptable salt thereof includethe following processes.

[0046] An analogy process to biosynthesis in which3-(3-benzyloxy-4-methoxyphenyl)-2-propenal is used as a startingmaterial [Chemical & Pharmaceutical Bulletin, 28(10), 3013 (1980)].

[0047] A process in whichsynthon-1-(3-benzyloxy-4-methoxyphenyl)-5-dimethylamino-1,4-pentadiene-3-one and dimethyl 3-oxoglutarate are used as startingmaterials followed by aromatic anelation and debenzylation [Chemical &Pharmaceutical Bulletin, 31(12), 4360 (1983)].

[0048] A process in which N,N-diethyl-2-methylbenzamide is subjected tolithiumization followed by condensation with an aromatic aldehyde tosubject to basic hydrolysis [Journal of Organic Chemistry, 49(5), 742(1984)]. A process in which copper chloride is used as a catalyst and asite-directed oxidative lactonising reaction is applied [Chemical &Pharmaceutical Bulletin, 44(10), 1890 (1996)].

[0049] A process starting from the treatment of orthotoluic acid withexcess amount of lithium isopropylamide [Synthetic Communications,26(9), 1753 (1996)].

[0050] A process in which orthotoluate is treated with lithiumisopropylamide to give an anion followed by polymerization [Synthesis,1, 72 (1980)]. A process in which a homophthalic acid derivative isheated with an aromatic acyl chloride followed by subjecting to alkalinedecomposition [Chemical & Pharmaceutical Bulletin, 29(9), 2491 (1981)].

[0051] A process in which homophthalic acid and benzoylchloride bromideare used as starting materials then alkaline hydrolyzed and via reducedracemic hydroxy acid [Indian Journal of Heterocyclic Chemistry, 8, 99(1998)].

[0052] A process achieved by heating benzocyclobutenone with alcohol[Tetrahedron Letters, 38(33), 5745 (1997)]. A process in which areaction between 4,5,6,7-tetraiodophthalic anhydride and phenyl acetateis executed [Egypt Journal of Chemistry, 22(2), 135 (1979)]

[0053] A process in which 3,4-dimethoxyphenyl acetate and phthalicanhydride are heated in the presence of sodium acetate followed bylactonization, and further, hydrolysis is executed in the presence ofaqueous tetrahydrofuran and tetrabutylammonium bromide [Journal ofOrganic Chemistry, 59(26), 8220 (1994)]

[0054] A process in which an alkylbenzoic acid derivative is cyclizedwith a palladium catalyst in the presence of benzoquinone [Chemical &Pharmaceutical Bulletin, 42(8), 1700 (1994)]

[0055] A process in which 3,5-disubstituted 4-hydroxybenzaldehyde, whichwas synthesized by treating 2,6-disubstituted phenol withhexamethylenetetramide and boric acid followed by hydrolysis withsulfuric acid, is used as a starting material to subject to Wittigreaction with phospholan generated from triphenylphosphonium bromide[European Journal of Medicinal Chemistry, 13(5), 425 (1978)].

[0056] Examples of the natural product which originally contains thecompound (I) or a pharmaceutically acceptable salt thereof includeplants and herbal medicines such as Hydrangea macrophylla Ser. var.Thunbergii MAKINO, Hydrangea serrata var. Thunbergii and HydrangeaeDulcis Folium. Examples of the plant which contains hydrangenol includeplants in genus hydrangea such as Hydrangea macrophylla Ser. andHydrangea macrophylla Ser. var. otaksa Makino, Dichroae radix and thelike. Improved plants so that plenty of compound (I) or apharmaceutically acceptable salt thereof is included, or cultured planttissues may also be used. The compound (I) or a pharmaceuticallyacceptable salt thereof is obtained from the extract of the plant bodyin the form of the sole material or the mixture.

[0057] For the purification from a natural product, a techniquegenerally used in the purification of compounds having the lowermolecular weight is employed in which the target compound is extractedusing an organic solvent followed by a solvent fractionation method, acolumn chromatographic method, an HPLC method, a recrystallizationmethod or the like.

[0058] Examples of the method of extraction include extraction withvarious solvents, supercritical fluid extraction and the like. Extractsmay further be treated by various methods of solid-liquid separationsuch as sedimentation, cake filtration, clear filtration, centrifugalfiltration, centrifugal sedimentation, separation by compression andfilter press, various concentration methods, various drying methods,methods of making various preparations such as granulation andpulverization, various purification methods, and the like.

[0059] Examples of the purification method include solvent fractionationmethods, column chromatographic methods, recrystallization methods andthe like. Specifically preferred is column chromatographic method usingvarious carriers such as DIAION HP-20 (manufactured by MitsubishiChemical Corporation) and Sephadex LH-20 (manufactured by Pharmacia).

[0060] Examples of the concentration and drying method include filmdrying methods such as freeze-drying, natural drying, hot air-drying,blow-drying, spray drying, drying under reduced pressure, sun-drying,vacuum drying, fluidized-bed drying, foam-bed drying and drum drying,drying methods such as ultrasonic drying and electromagnetic wavedrying. Preferred are a spray drying method and a freeze-drying method.

[0061] In the step of extraction and treatment of an extract, anantioxidant, a preservative, etc. may be added.

[0062] As the solvent used in extraction with a solvent, any solventwhich can extract the compound (I) or a pharmaceutically acceptable saltthereof can be used. Examples of the preferred solvent include aqueousmedia such as water, distilled water, deionized water, an aqueoussolution of an inorganic salt and buffer, monovalent alcohols such asmethanol, ethanol, propanol and butanol, polyvalent alcohols such aspropylene glycol and glycerol, and organic solvents such as hexane,toluene, petroleum ether, benzene, ethyl acetate, chloroform,dichloromethane, 1,1,2-trichloroethene, dimethyl sulfoxide and acetone.Preferred are aqueous media and alcohols.

[0063] Examples of the buffer include phosphate buffer, citrate bufferand the like. Examples of the inorganic salt for the aqueous solution ofan inorganic salt include sodium chloride, potassium chloride, calciumchloride and the like.

[0064] Preferred alcohols are monovalent alcohols and a preferredmonovalent alcohol is ethanol.

[0065] These solvents can be used alone or as a mixture of multiplesolvents. As the mixed solvent, water-containing alcohols are preferred.Water-containing monovalent alcohols are more preferred andwater-containing ethanol is particularly preferred. The water content ispreferably 70′ or lower, and more preferably 40% or lower.

[0066] As the solvent, supercritical fluidized carbon dioxide may alsobe employed.

[0067] Extraction is conducted using the solvent in an amount of 0.1part by weight to 10000 parts by weight, and preferably 1 part by weightto 100 parts by weight per 1 part by weight of a plant body. Althoughthe temperature for extraction is not particularly limited, it ispreferably 0° C. to 100° C., and more preferably 20° C. to 90° C.Although the time period for extraction is not particularly limited, itis preferably one minute to one week, and more preferably 30 minutes toone day.

[0068] Concentration of the extract may be conducted under either normalpressure or reduced pressure. Although the temperature for theconcentration is also not particularly limited, it is preferably 40° C.or less. The compound (I) or a pharmaceutically acceptable salt thereofmay be used in the form of a mixture of the extract, however, it may bealso isolated as needed. The isolation can be performed throughextraction with an organic solvent such as lower alkyl ketone, loweralcohol, lower fatty acid, lower fatty acid ester, lower aliphaticether, halogen-substituted or unsubstituted lower hydrocarbon, or thelike followed by subjecting the concentrate of this extract to columnchromatography using silica gel, alumina or the like as a carrier.

[0069] Examples of the lower alkyl ketone include acetone,methylethylketone and the like. Examples of lower alcohol includemethanol, ethanol and the like. Examples of the lower fatty acid includeacetic acid and the like. Examples of the lower fatty acid ester includeethyl ether, isopropyl ether and the like. Examples of thehalogen-substituted or unsubstituted lower hydrocarbon includechloroform, carbon tetrachloride, n-hexane and the like. These may beused alone or as a mixture.

[0070] In the present invention, protection of a liver function involvesactivities to protect the liver functions from various disorders,activities to prevent the liver functions from a disorder, and the like.Improvement of a liver function involves activities to recover or curethe disordered functions of liver, activities to improve or enhanceliver function, and the like.

[0071] The term “liver function” as used herein means every function ofthe liver and there is no limit as to the definition of the term.Specific examples of the liver function include those relating to bloodand circulation such as storage of blood (adjustment of the amount ofcirculating blood), treatment of blood pigments (discharge ofhemoglobin), formation of bile, enterohepatic circulation of bilepigments, and synthesis of plasma proteins (e.g. acute phase proteins,albumin, blood coagulation factors, steroid-binding proteins and otherhormone-binding proteins), metabolic functions such as metabolism ofnutrients and vitamins (e.g. glucose and other sugars, amino acids,lipids-fatty acids, cholesterol, lipoproteins, lipid-soluble vitaminsand water-soluble vitamins), detoxification or decomposition functionssuch as inactivation of various substances (e.g. alcohols, acetaldehyde,toxins, steroids such as estrogen and androsterone, and other hormones),immune functions [“Seirigaku Tenbo” (View of Physiology), 19th edition(Mar. 31, 2000), “Atarashii Rinsho Eiyogaku” (New Study of ClinicalNutrition), 3rd revision (May 20, 2000)], and the like. These functionsall suffer damage from an alcohol.

[0072] The liver function protecting or improving agent of the presentinvention can improve a liver function disorder through theadministration thereof to a human or an animal already having a disorderin liver function. Further, the liver function protecting or improvingagent of the present invention can prevent a liver function disorderthrough the administration thereof to a human or an animal with nomanifestation of a liver function disorder.

[0073] The liver function protecting or improving agent of the presentinvention contains the compound (I) or a pharmaceutically acceptablesalt thereof, and may contain one or more pharmaceutically acceptablecarriers as needed, as well as an active ingredient for the therapy asneeded.

[0074] The liver function protecting or improving agent of the presentinvention is produced by mixing the compound (I) or a pharmaceuticallyacceptable salt thereof with a carrier as needed, according to anarbitrary method which is well known in the technical field ofpharmaceutics.

[0075] It is desirable to employ the most effective route foradministration of the formulation for treatment. Examples of suitableadministration route include oral administration and parenteraladministration such as intravenous administration, intraperitonealadministration and subcutaneous administration. Among these, oraladministration is preferred.

[0076] Examples of dosage form for the administration include tablets,powders, granules, pills, suspensions, emulsions, infusa, capsules,syrups, injections, liquids, elixirs, extracts, tinctures, fluidextracts and the like.

[0077] Dosage forms suitable for oral administration for example,extracts, tinctures, fluid extracts and the like can be prepared byextracting the compound (I) or a pharmaceutically acceptable saltthereof from a body of e.g., a plant of the family Saxifragaceae withfor example, water, ethanol or a mixture of water and ethanol, with orwithout the following concentration of the extract.

[0078] Liquid preparations suitable for oral administration such assyrups can be prepared using carriers such as water, sugars (e.g.sucrose, sorbitol and fructose), glycols (e.g. polyethylene glycol andpropylene glycol), oils (e.g. sesame oil, olive oil and soybean oil),antiseptics (e.g. p-hydroxybenzoic acid esters), paraoxybenzoic acidderivatives (e.g. methyl paraoxybenzoate), preservatives (e.g. sodiumbenzoate) and flavors (e.g. strawberry flavor and peppermint).

[0079] Dosage forms suitable for oral administration for example,tablets, powders, granules and the like can be prepared using sugarssuch as lactose, white sugar, glucose, sucrose, mannitol and sorbitol,starch such as potato starch, wheat starch and corn starch, inorganicsubstances such as calcium carbonate, calcium sulfate, sodiumbicarbonate and sodium chloride, excipients such as crystallinecellulose and plant powders (e.g. licorice powder and gentian powder),disintegrating agents such as starch, agar, gelatin powder, crystallinecellulose, carmellose sodium, carmellose calcium, calcium carbonate,sodium bicarbonate and sodium alginate, lubricants such as magnesiumstearate, talc, hydrogenated vegetable oil, macrogol and silicone oil,binders such as polyvinyl alcohol, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, carmellose, gelatin and starch paste,surfactants such as fatty acid ester, plasticizers such as glycerin, andthe like.

[0080] Preparations suitable for parenteral administration such asinjections, preferably comprise a sterilized aqueous agent containing anactive compound which is isotonic to the recipient's blood. In the caseof an injection, for example, an injectable solution is prepared using acarrier such as a salt solution, a glucose solution, or a mixture of asalt solution and a glucose solution.

[0081] The antiseptics, preservatives, surfactants, etc. as describedabove can also be employed in such preparations for parenteraladministration.

[0082] The dose and dosage frequency of the liver function protecting orimproving agent of the invention will vary depending on theadministration route, the age and body weight of a patient, and propertyor severity of the symptom to be treated, without specific restriction.In general, when orally administered to an adult, it is suitable toadminister in an amount of 0.01 mg to 50 g, preferably 0.05 mg to 10 gin terms of the compound (I) or a pharmaceutically acceptable saltthereof once to several times per day. In the case of parenteraladministration such as intravenous administration, it is suitable toadminister to an adult in an amount of 0.001 mg to 50 g, preferably 0.01mg to 10 g in terms of the compound (I) or a pharmaceutically acceptablesalt thereof once to several times per day. In the case ofadministration to an animal, the dose and dosage frequency will varydepending on the age and kind of the animal, and the property orseverity of the symptom, without specific restriction. In general, whenorally administered, it is suitable to administer in an amount of 0.1 μgto 10 g, preferably 1 μg to 1 g per kg of body weight once to severaltimes per day. In the case of parenteral administration such asintravenous administration, it is suitable to administer in an amount of0.01 μg to 10 g, preferably 0.1 μg to 1 g per kg of body weight once toseveral times per day. However, the dose and dosage frequency may varydepending upon the above-mentioned various conditions.

[0083] The food and drink or the feed in which the compound (I) or apharmaceutically acceptable salt thereof is added may include thoseprepared by adding the compound (I) or a pharmaceutically acceptablesalt thereof to a food and drink or a feed which originally contains thecompound (I) or a pharmaceutically acceptable salt thereof or which doesnot originally contain the compound (I) or a pharmaceutically acceptablesalt thereof to produce in a process for producing ordinary foods anddrinks or feeds. Further, the food and drink or the feed may beprocessed by a method of processing ordinary foods and drinks or feeds.Examples of the method of processing include granulating methods such asfluidized bed granulation, stirring granulation, extrusion granulation,rolling granulation, air stream granulation, compression moldinggranulation, disruption granulation, spray granulation and blastinggranulation, coating methods such as pan coating, fluidized bed coatingand dry coating, swelling methods such as puff drying, excess steammethod, foam mat method and microwave heating method, and the like.

[0084] The food and drink or the feed containing the compound (I) or apharmaceutically acceptable salt thereof or materials therefor are notparticularly limited, which may include those comprising the compound(I) or a pharmaceutically acceptable salt thereof, and those which arenot substantially comprising the compound (I) or a pharmaceuticallyacceptable salt thereof.

[0085] The liver function protecting or improving activity of the foodand drink or the feed which comprises the compound (I) or apharmaceutically acceptable salt thereof can be enhanced by addingthereto the compound (I) or a pharmaceutically acceptable salt thereof.

[0086] Although the amount of the compound (I) or a pharmaceuticallyacceptable salt thereof of the invention to be added to the food anddrink or the feed is not particularly limited as long as it is an amountto give a content which enables the food and drink or the feed toexhibit liver function protecting or improving activity, the content ofthe compound (I) or a pharmaceutically acceptable salt thereof allowedin the food and drink or the feed is preferably 0.001 to 100%, morepreferably 0.01 to 100%, and particularly preferably 0.1 to 100%.

[0087] Specific examples of the food and drink to which the compound (I)or a pharmaceutically acceptable salt thereof is added include juice,soft drinks, soup, tea, dairy products (e.g. lactic acid bacteriabeverages, fermented milk, frozen dessert, butter, cheese, yogurt,processed milk and skim milk powder), meat products (e.g. ham, sausageand hamburger), fish cake products, egg products (e.g. fried or steamedfoods made of beaten eggs), confectionery (e.g. cookies, jelly, snacksand chewing gum), bread, noodles, pickles, smoked foods, dry foods,preserved foods boiled in soy sauce, seasonings and the like.

[0088] The food and drink may be in any of the forms such as a powderfood, a sheet-shaped food, a bottled food, a canned food, a retortpouched food, a capsule food, a tablet-shaped food, a liquid food and ahealth drink, and the like.

[0089] The food and drink of the present invention is used for theprotection or improvement of liver function, as a health food and drinkor a functional food and drink.

[0090] When the present food and drink for protecting or improving liverfunction which comprises the compound (I) or a pharmaceuticallyacceptable salt thereof as an active ingredient is ingested, the amountof intake is not particularly limited, however, it is 0.01 mg to 50 g,and preferably 0.05 mg to 10 g in terms of the weight of the compound(I) or a pharmaceutically acceptable salt thereof given to an adult perday. It is ingested in this amount of intake for 1 day to 1 year, andpreferably for 2 weeks to 3 months. However, this amount of intake ismerely a typical example, and can be appropriately adjusted to fallwithin a suitable range according to the recipient's extent of thesymptom, age, weight, etc.

[0091] Examples of the feed to which the compound (I) or apharmaceutically acceptable salt thereof is added include feeds foranimals such as mammals, birds, reptiles, amphibians and fish. Specificexamples of the feed include feeds for pets such as dogs, cats and mice,feeds for livestock such as cows and pigs, feeds for poultry such ashens and turkeys, feeds for cultivated fish such as sea breams and youngyellowtails, and the like.

[0092] The feed of the present invention can be produced byappropriately mixing the compound (I) or a pharmaceutically acceptablesalt thereof with a feed material.

[0093] Exemplary feed materials include grains, bran, vegetable oilcakes, animal feed materials, other feed materials, purified productsand the like.

[0094] Examples of the grain include milo, wheat, barley, oats, rye,nonglutinous brown rice, buckwheat, foxtail millet, broomcorn millet,Japanese millet, corn, soybean and the like.

[0095] Examples of the bran include rice bran, defatted rice bran, wheatbran, wheat middlings, wheat germ, barley bran, pellet, corn bran, corngerm and the like.

[0096] Examples of the vegetable oil cake include soybean oil cake,soybean flower, linseed oil cake, cottonseed oil cake, peanut oil cake,safflower oil cake, coconut oil cake, palm oil cake, sesame oil cake,sunflower oil cake, rapeseed oil cake, kapok oil cake, mustard seed oilcake and the like.

[0097] Examples of the animal feed material include fish meal (e.g.northern ocean meal, imported meal, whole meal and coastal meal), fishsoluble, meat meal, meat and bone meal, blood powder, degraded hair,bone meal, treated by-products for livestock, feather meal, silkwormpupa, skim milk powder, casein, dry whey and the like.

[0098] Examples of other feed material include stalks and leaves ofplants (e.g. alfalfa, hay cube, alfalfa leaf meal and powder of falseacacia), processed industrial by-products of corn (e.g. corn gluten,meal, corn gluten feed and corn steep liquor), processed starch products(e.g. starch), sugar, fermentation industrial products (e.g. yeast, beercake, malt root, alcohol cake and soy sauce cake), agriculturalby-products (e.g. processed citrus fruit cake, tofu cake, coffee cakeand cocoa cake) and others (e.g. cassava, broad bean, guar meal,seaweeds krill, spirulina, chlorella and minerals) and the like.

[0099] Examples of the purified product include proteins (e.g. caseinand albumin), amino acids, carbohydrates (e.g. starch, cellulose,sucrose and glucose), minerals, vitamins and the like.

[0100] When the feed to which the compound (I) or a pharmaceuticallyacceptable salt thereof is added is fed to an animal, the amount ofintake is not particularly limited, however, it is 0.1 μg to 10 g, andpreferably 0.1 μg to 1 g in terms of the weight of the compound (I) or apharmaceutically acceptable salt thereof per kg of the body weight ofthe animal per day. It is ingested in this amount of intake for 1 day to1 year, and preferably for 2 weeks to 3 months. However, this amount ofintake is merely a typical example, and can be appropriately adjusted tofall within a suitable range according to the kind, age, body weight,etc. of an animal to be fed.

[0101] The additive for foods and drinks or feeds in which the compound(I) or a pharmaceutically acceptable salt thereof is added may beprepared by adding the compound (I) or a pharmaceutically acceptablesalt thereof to an additive for foods and drinks or feeds, optionallyfollowed by adding an additive generally employed in foods and drinks orfeeds as needed, for example, additives listed in Food AdditivesIndication Pocket Book (Japan Food Additives Association, Jan. 6, 1997)such as sweeteners, coloring agents, preservatives, thickeningstabilizers, antioxidants, color developing agents bleaching agents,fungicides, gum bases, bitter agents, enzymes or enzyme sources, glossagents, sour agents, seasonings, emulsifiers, fortifier dietarysupplements, additional materials for preparation, flavors, spiceextracts and the like. Moreover, the carriers illustrated in the abovedescription of the liver function protecting or improving agent may alsobe added.

[0102] Although concentration of the compound (I) or a pharmaceuticallyacceptable salt thereof in the additive for foods and drinks or feeds isnot particularly limited, it is preferably 1 to 99%, more preferably 10to 90%, particularly preferably 20 to 80%.

[0103] Examples of the sweetener include aspartame, licorice, stevia,xylose and Momordica grosvenori and the like.

[0104] Examples of the coloring agent include carotenoid, turmericpigment, flavonoid, caramel pigment, oriental gromurell pigment,spirulina pigment, chlorophyll, red sweet potato pigment, red Chineseyam pigment, perilla pigment, blueberry pigment and the like.

[0105] Examples of the preservative include sodium sulfite, benzoic acidand benzoates, extract of Aralia cordata, Japanese Styrax benzoinextract, Rumpet roman extract, sorbic acid and sorbates, propionic acidand propionates, and the like.

[0106] Examples of the thickening stabilizer include gums such as gumarabic and xanthane gum, alginic acid and alginates, chitin, chitosan,aloe extract, guar gum, hydroxypropyl cellulose, casein sodium,cornstarch, carboxymethylcellulose, gelatin, agar, dextrin, methylcellulose, polyvinyl alcohol, microfibrous cellulose, microcrystallinecellulose, seaweed cellulose, sodium polyacrylate, sodium polyphosphate,carrageenan, yeast cell wall, extract of konjac, nata de coco, mannanand the like.

[0107] Examples of the antioxidant include vitamin C, sodiumethylenediaminetetraacetate, calcium ethylenediaminetetraacetate,erythorbic acid, oryzanol, catechin, quercetin, clove extract,enzyme-treated rutin, apple extract, sesame oil extract,dibutylhydroxytoluene, fennel extract, horseradish extract, waterdropwort extract, tea extract, Tempeh extract, extract of Houttuyniacordata, tocotrienol, tocopherols, rapeseed oil extract, green coffeeextract, sunflower seed, ferulic acid, butylhydroxyanisole, blueberryleaf extract, propolis extract, hego-ginkgo leave extract, hesperetin,pepper extract, garden balsam extract, gallic acid, myrica extract,eucalyptus extract, rosemary extract and the like.

[0108] Examples of the color developing agent include sodium nitrite andthe like, and examples of the bleaching agent include sodium sulfite andthe like.

[0109] Examples of the fungicide include orthophenylphenol and the like.

[0110] Examples of the gum base include methyl acetylricinoleate,Japanese lacquer wax, ester gum, elemi resin, urucury wax, ozokerite,opopanax resin, kauri gum, carnauba wax, guaiacum resin, gutta katiau,gutta hangkang, gutta percha, glycerin fatty acid ester, spermaceti wax,copaiba balsam, copal resin, gum, rice bran wax, sugar cane wax,shellac, jelutong, sucrose fatty acid ester, sorba, sorbitan fatty acidester, talc, calcium carbonate, dammar resin, chicle, chilte, tunu, lowmolecular weight gum, paraffin wax, fir balsam, propylene glycol fattyacid ester, powdered pulp, powdered rice husks, jojoba wax,polyisobutylene, polybutene, microcrystalline wax, mastic, massarandubachocolate, beeswax, calcium phosphate and the like.

[0111] Examples of the bitter agent include isoalpha bitter acid,caffeine, kawaratake extract, cinchona extract, Amur cork extract,gentian extract, spice extracts, enzyme-treated naringin, Jamaicaquassia extract, theobromine, naringin, bitter ash extract, warmwoodextract, isodonis extract, himematsutake extract, borapet, methylthioadenosine, litchi extract, olive tea, sour orange extract, hopextract, mugwort extract and the like.

[0112] Examples of the enzyme or enzyme source include amylase, trypsin,rennet, lactic acid bacteria and the like.

[0113] Examples of the gloss agent include Japanese lacquer wax,vegetable wax and the like.

[0114] Examples of the sour agent are adipic acid, itaconic acid, citricacid and citrates, succinic acid and succinates, sodium acetate,tartaric acid and tartrates, carbon dioxide, lactic acid, phytic acid,fumaric acid, malic acid, phosphoric acid and the like.

[0115] Examples of the seasoning include amino acids such as asparagine,aspartic acids, glutamic acid, glutamine, alanine, isoleucine, glycine,serine, cystine, tyrosine, leucine and proline, nucleic acids such assodium inosinate, sodium uridylate, sodium guanylate, sodium cytidylate,calcium ribonucleotide and sodium ribonucleotide, organic acids such ascitric acid and succinic acid, potassium chloride, sodium solution oflow salt content prepared from salt lake water, crude potassium chloridefrom sea water, whey salt, tripotassium phosphate, dipotassiumhydrogenphosphate, potassium dihydrogenphosphate, disodiumhydrogenphosphate, sodium dihydrogenphosphate, trisodium phosphate,chlorella extract and the like.

[0116] Examples of the emulsifier include fatty acid monoglyceride,sorbitan fatty acid ester and the like.

[0117] Examples of the fortifier dietary supplement include zinc salts,vitamin C, various amino acids, 5-adenylic acid, iron chloride,hesperidin, various kinds of burnt calcium, various kinds of unburntcalcium, dibenzoylthiamine, calcium hydroxide, calcium carbonate,thiamine hydrochloride, dunaliella carotene, tocopherol, nicotinic acid,carrot carotene, palm oil carotene, calcium pantothenate, vitamin A,hydroxyproline, calcium dihydrogenpyrophosphate, iron (II)pyrophosphate, iron (III) pyrophosphate, ferritin, heme iron,menaquinone, folic acid, riboflavin and the like.

[0118] Examples of the additional material for preparation includeprocessing aids such as acetone and ion exchange resin, extract of figleaf, extract of rice straw ash, kaolin, glycerin fatty acid ester,mulberry extract, bone ash, perilla extract, ginger extract, varioustannins, Phaffia color extract, grape seed extract, ethanol and thelike.

[0119] Examples of the flavor include strawberry flavor, peppermintflavor and the like.

[0120] Examples of the spice extract include chili pepper extract,garlic extract and the like.

[0121] The present invention is hereinafter explained in detailaccording to Examples. However, the Examples described below should notbe construed as limiting the invention. Equipments used in the Examplesare as those described below.

[0122] Mass spectrometer (device: JMS-HX110/110A (JEOL, Ltd.), FAB gas:Xe, charged voltage: 10 kV, matrix: m-NBA), high resolution FAB massspectrum (positive mode; matrix: m-NBA): nuclear magnetism resonancemethod (device: JNM-A400 (JEOL, ¹H—NMR: 400 MHz, ¹³C—NMR: 100 MHz).

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Isolation ofPhyllodulcin

[0123] Dry powder of Hydrangeae Dulcis Folium (manufactured by ShihiraShoten) in an amount of 1.0 kg was extracted twice with 20 L of methanolat room temperature through leaving to stand for 2 days. Thus obtainedextract was concentrated and evaporated to dryness to obtain 425.8 g ofa methanol extract. The extract was subjected to fractionation using 6 Lof a silica gel column (Wako gel C-300, manufactured by Wako PureChemical Industries, Ltd.) by a 10% stepwise method starting fromn-hexane to AMC solution (referring to a chloroform solution containing0.1% acetic acid and 0.5% of methanol).

[0124] Thus eluted 90% AMC solution-containing n-hexane fraction and100% AMC solution fraction (each fraction of 6 L) were mixed to obtain afraction I.

[0125] The fraction I was concentrated followed by subjecting tofractionation using 2 L of a silica gel column (Wako gel C-300,manufactured by Wako Pure Chemical Industries, Ltd.) by a 10% stepwisemethod starting from n-hexane to acetone to obtain 50%, 60% and 70%acetone-containing n-hexane fractions (each fraction of 2 L). The threefractions were combined and concentrated, and thus 26.2 g ofphyllodulcin powder was obtained by a recrystallization process.

[0126] The values of ¹H—NMR and ¹³C—NMR of the isolated compound eachagreed with values of ¹H-NMR and ¹³C-NMR of an authentic sample.

EXAMPLE 2 Isolation of Hydrangenol

[0127] Dry powder of Hydrangeae Dulcis Folium (manufactured by ShihiraShoten) in an amount of 1.0 kg was extracted twice with 20 L of methanolat room temperature through leaving to stand for 2 days. Thus obtainedextract was concentrated and evaporated to dryness to obtain 425.8 g ofa methanol extract. The extract was subjected to fractionation using 6 Lof a silica gel column (Wako gel C-300, manufactured by Wako PureChemical Industries, Ltd.) by a 10% stepwise method starting fromn-hexane to AMC solution, and then a 0.5% stepwise method starting fromchloroform to methanol.

[0128] Thus eluted 1.0% methanol-containing chloroform fraction (6 L)was combined to give a fraction II.

[0129] The fraction II was concentrated followed by subjecting tofractionation using 400 mL of a normal phase column (LiChroprep Si60,manufactured by Merck & Co., Inc.) by a 20% stepwise method startingfrom n-hexane to AMC solution, and then a 1.0% stepwise method startingfrom chloroform to methanol to obtain 60%, 80% and 100% AMCsolution-containing n-hexane fractions and a 1.0% methanol-containingchloroform fraction (each fraction of 2.4 L). The four fractions werecombined and concentrated, and thereafter subjected to fractionationusing 2 L of a silica gel column (Wako gel C-300) by a 5% stepwisemethod starting from toluene to ethyl acetate to obtain toluenefractions (each fraction of 2 L) each containing 15%, 20% and 25% ethylacetate. The three fractions were combined and concentrated, and thus18.4 g of hydrangenol powder was obtained by a recrystallizationprocess.

[0130] Values of H-NMR spectrum [400 MHz, heavy DMSO solution, 30° C.]and ¹³C-NMR spectrum [100 MHz, heavy DMSO solution, 30° C.] of theisolated hydrangenol agreed with values for hydrangenol in theliteratures [¹H—NMR: Agricultural Chemistry (Nougei Kagaku) Vol. 47, No.10, page 605, 1973, and ¹³C—NMR: Chemical & Pharmaceutical Bulletin,44(8), 1440 (1996)].

EXAMPLE 3 Isolation of Thunberginol A

[0131] Dry powder of Hydrangeae Dulcis Folium (manufactured by ShihiraShoten) in an amount of 1.0 kg was extracted twice with 20 L of methanolat room temperature through leaving to stand for 2 days. Thus obtainedextract was concentrated and evaporated to dryness to obtain 425.8 g ofa methanol extract. The extract was subjected to fractionation using 6 Lof a silica gel column (Wako gel C-300, manufactured by Wako PureChemical Industries, Ltd.) by a 10% stepwise method starting fromn-hexane to AMC solution, and then a 0.5% stepwise method starting fromchloroform to methanol.

[0132] Thus eluted 2.0% and 2.5% methanol-containing chloroformfractions (each fraction of 6 L) were combined to give a fraction III.

[0133] The fraction III was concentrated followed by subjecting tofractionation using 2 L of a silica gel column (Wako gel C-300,manufactured by Wako Pure Chemical Industries, Ltd.) by a 10% stepwisemethod starting from n-hexane to acetone to obtain a 50%acetone-containing n-hexane fraction (2 L). The fraction wasconcentrated followed by subjecting to fractionation using 100 mL of anormal phase column (LiChroprep Si60, manufactured by Merck & Co., Inc.)by a 5% stepwise method starting from toluene to ethyl acetate to obtaintoluene fractions (each fraction of 600 mL) each containing 10%, 15% and20% ethyl acetate. The three fractions were combined and concentratedfollowed by subjecting to fractionation using 100 mL of a reverse phasecolumn (Cosmosil140C₁₈OPN, manufactured by Nacalai Tesque, Inc.) by a10% stepwise method starting from water to methanol to obtain 50%, 60%and 70% methanol-containing water fractions (each fraction of 600 ml).The three fractions were combined and concentrated, and thus 0.6 g ofthunberginol A was obtained by a recrystallization process.

[0134] Values of ¹H—NMR spectrum [400 MHz, heavy DMSO solution, 30° C.]and ¹³C—NMR spectrum [100 MHz, heavy DMSO solution, 30° C.] of theisolated thunberginol A agreed well with values in the literatures[Chem. Pharm. Bull., 42(11), 2225 (1994)].

EXAMPLE 4 Production of a Feed Containing Phyllodulcin at aConcentration of 0.366%

[0135] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 39.734 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 1  0.366 wt %

EXAMPLE 5 Production of a Feed Containing Phyllodulcin at aConcentration of 0.122%

[0136] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 39.978 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 1  0.122 wt %

EXAMPLE 6 Production of a Feed Containing Phyllodulcin at aConcentration of 0.0366%

[0137] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)   20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.0634 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)   25.0 wt % Powderproduced in Example 1  0.0366 wt %

EXAMPLE 7 Production of a Feed Containing Hydrangenol at a Concentrationof 0.324%

[0138] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 39.776 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 2  0.324 wt %

EXAMPLE 8 Production of a Feed Containing Hydrangenol at a Concentrationof 0.108%

[0139] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 39.992 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 2  0.108 wt %

EXAMPLE 9 Production of a Feed Containing Hydrangenol at a Concentrationof 0.0324%

[0140] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)   20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.0676 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)   25.0 wt % Powderproduced in Example 2  0.0324 wt %

EXAMPLE 10 Production of a Feed Containing Thunberginol A at aConcentration of 0.039%

[0141] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.061 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 3  0.039 wt %

EXAMPLE 11 Production of a Feed Containing Thunberginol A at aConcentration of 0.013%

[0142] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)  20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.087 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)  25.0 wt % Powderproduced in Example 3  0.013 wt %

EXAMPLE 12 Production of a Feed Containing Thunberginol A at aConcentration of 0.0039%

[0143] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.)   20.0 wt % Corn oil (Nacalai Tesque, Inc.)  5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)   0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.0961 wt % AIN-76vitamin (Oriental Yeast Co., Ltd.)   1.0 wt % AIN-76 mineral (OrientalYeast Co., Ltd.)   3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)   5.0wt % Casein (Wako Pure Chemical Industries, Ltd.)   25.0 wt % Powderproduced in Example 3  0.0039 wt %

COMPARATIVE EXAMPLE 1

[0144] Following materials were admixed to produce a feed. Sucrose(Kishida Chemical Co., Ltd.) 20.0 wt % Corn oil (Nacalai Tesque, Inc.) 5.0 wt % Choline bitartrate (Tokyo Kasei Kogyo Co., Ltd.)  0.4 wt %Corn starch (Nippon Starch Chemical Co., Ltd.) 40.1 wt % AIN-76 vitamin(Oriental Yeast Co., Ltd.)  1.0 wt % AIN-76 mineral (Oriental Yeast Co.,Ltd.)  3.5 wt % Cellulose (Oriental Yeast Co., Ltd.)  5.0 wt % Casein(Wako Pure Chemical Industries, Ltd.) 25.0 wt %

EXAMPLE 13 Suppression of Galactosamine-induced Rat Hepatopathy byPhyllodulcin and Hydrangenol.

[0145] Male Wistar white rats (150±20 g, purchased from Japan SLC) werekept for at least 3 days under a fixed condition (temperature: 24±2° C.,humidity: 60±5%, light and dark interval: 12 hours) for adaptation, andthen fed respectively with the feeds produced in Examples 4-8 and thefeed produced in Comparative Example 1 for 15 days. On the 14th day, 350mg/kg of galactosamine (dissolved in physiological saline at aconcentration of 35 mg/ml, and adjusted to pH 7.1) was intraperitoneallyadministered to each rat. Twenty-two hours after the administration ofgalactosamine, each of the rats was subjected to laparotomy underanesthesia with Nembutal and blood was collected.

[0146] Using thus obtained blood samples, blood GPT activity wasmeasured as an indication of a liver function in the following manner.The collected blood was coagulated and separated by centrifugation toobtain a serum. The GPT level in the serum was measured using FujiDrychem System 3500 (manufactured by Fuji Photo Film Co., Ltd.) withthus obtained serum. The GPT activity of each test group was calculatedas the relative value (%) from the value obtained for each Example basedon the value obtained for the feed in Comparative Example 1 expressed as100%. The value is expressed in terms of average value±standard errorand the statistical test of significance was carried out by Student'sT-test.

[0147] The results are shown in Table 2. TABLE 2 Active ConcentrationGPT activity Test of ingredient Feed (%) (%) significance phyllodulcinExample 4 0.366 11.8 ± 18.0 p = 0.0002 Example 5 0.122 54.7 ± 32.2 p =0.0400 Example 6 0.037 52.7 ± 59.9 p = 0.1241 hydrangenol Example 70.324  64.6 ± 116.5 p = 0.5177 Example 8 0.108 27.8 ± 45.4 p = 0.0343

[0148] When the feeds of Examples 4-8 were administered, the GPTactivity in serum which is an indication of a liver function disorderwas kept low in comparison with the case in which the feed ofComparative Example was administered. It is revealed that hepatopathywas suppressed accordingly.

[0149] During 15 days of the feeding, there was no difference in weightincrease among the cases where any of the feeds was given, and noabnormality was also recognized in appearance and action.

EXAMPLE 14 Production of a Compound Formulation Containing Phyllodulcin

[0150] A liver function protecting or improving agent was produced bymixing the following composition. Phyllodulcin produced in Example 1 4.9 g Pine-dex #3  4.9 g Iron (III) pyrophosphate (iron source; KokusanChemical 0.01 g Co., Ltd.) Phoscal EFC (calcium source; Nikko FineProducts)  0.1 g Vitamin Mix (Merck & Co., Inc.)  0.1 g

EXAMPLE 15 Production of a Compound Formulation Containing Hydrangenol

[0151] A liver function protecting or improving agent was produced bymixing the following composition. Hydrangenol produced in Example 2  4.9g Pine-dex #3  4.9 g Iron (III) pyrophosphate (iron source; KokusanChemical 0.01 g Co., Ltd.) Phoscal EFC (calcium source; Nikko FineProducts)  0.1 g Vitamin Mix (Merck & Co., Inc.)  0.1 g

EXAMPLE 16 Production of a Compound Formulation Containing ThunberginolA

[0152] A liver function protecting or improving agent was produced bymixing the following composition. Thunberginol A produced in Example 3 0.49 g Pine-dex #3  0.49 g Iron (III) pyrophosphate (iron source;Kokusan Chemical 0.001 g Co., Ltd.) Phoscal EFC (calcium source; NikkoFine Products)  0.01 g Vitamin Mix (Merck & Co., Inc.)  0.01 g

EXAMPLE 17

[0153] The liver function protecting or improving agent produced inExample 16 in an amount of 1 g was dispersed into 10 ml of water toproduce a drink for protecting or improving a liver function.

EXAMPLE 18 Production of a Cake Including Phyllodulcin

[0154] Cookies (30 pieces) were prepared from the following ingredients.Soft flour 100 g Starch 74 g Water 14 g Phyllodulcin produced in Example1 3 g Baking powder 2 Tsp. Salt 2 Tsp. Egg 1 Butter 80 g Milk 2 Tbsp.

INDUSTRIAL APPLICABILITY

[0155] According to the present invention, a liver function protectingor improving agent, a food and drink or a feed for protecting orimproving liver functions, and an additive for foods and drinks or anadditive for feeds having a liver function protecting or improvingactivity can be provided.

1. A liver function protecting or improving agent which comprises acompound represented by the formula (I)

{in the formula (I), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ may be thesame or different, and represent hydrogen, halogen, hydroxy, alkoxy oralkyl; and R^(A) represents the formula (II)

[in the formula (II), wherein R¹⁰ and R¹¹ may be the same or different,and represent hydrogen or halogen, or R¹⁰ and R¹¹ together represent abinding] or the formula (III)

[in the formula (III), R¹² represents hydrogen, halogen, hydroxy,alkoxy, cyano or alkyl, R¹³ and R¹⁴ may be the same or different, andrepresent hydrogen or halogen, or R¹³ and R¹⁴ together represent abinding]} or a glycoside thereof [hereinafter referred to as compound(I)] or a pharmaceutically acceptable salt thereof.
 2. The liverfunction protecting or improving agent according to claim 1, wherein R¹,R³, R⁵, R⁸ and R⁹ represent hydrogen.
 3. The liver function protectingor improving agent according to claim 1 or 2, wherein R² representshydrogen or hydroxy, R⁴ represents hydroxy, R⁶ and R⁷ may be the same ordifferent, and represent hydrogen, hydroxy or alkoxy.
 4. The liverfunction protecting or improving agent according to any one of claims 1to 3, wherein R^(A) represents the formula (II).
 5. The liver functionprotecting or improving agent according to any one of claims 1 to 3,wherein R^(A) represents the formula (III).
 6. The liver functionprotecting or improving agent according to claim 1, wherein the compound(I) is phyllodulcin.
 7. The liver function protecting or improving agentaccording to claim 1, wherein the compound (I) is hydrangenol.
 8. A foodand drink to which the liver function protecting or improving agentaccording to any one of claims 1 to 7 is added.
 9. A feed to which theliver function protecting or improving agent according to any one ofclaims 1 to 7 is added.
 10. An additive for foods to which the liverfunction protecting or improving agent according to any one of claims 1to 7 is added.
 11. A feed additive to which the liver functionprotecting or improving agent according to any one of claims 1 to 7 isadded.
 12. The liver function protecting or improving agent according toany one of claims 1 to 7, which is administered orally.
 13. The liverfunction protecting or improving agent according to any one of claims 1to 7, wherein the liver function is a function affected by an alcohol.14. A method for protecting or improving a liver function whichcomprises administering a therapeutically effective amount of thecompound (I) or a pharmaceutically acceptable salt thereof to an animalincluding human.
 15. Use of the compound (I) or a pharmaceuticallyacceptable salt thereof for manufacturing a liver function protecting orimproving agent.